Fructans are a type of water-soluble carbohydrate whose primary function is to provide a readily accessible energy reserve for plant growth. Fructans are associated with various advantageous characters in grasses, such as cold and drought tolerance, increased tiller survival, enhanced persistence, good regrowth after cutting or grazing, improved recovery from stress, early spring growth and increased nutritional quality.
Fructan synthesis and metabolism in grasses and cereals is complex. Fructans consist of linear or branched fructose chains attached to sucrose. The chain length of plant fructans ranges from three up to a few hundred fructose units. Different types of fructans can be distinguished based on the linkage types present. In perennial ryegrass three types of fructans have been identified: inulins, inulin neoseries and levan neoseries, with four fructosyltransferse (FT) enzymes involved in this fructan profile.
The enzyme 1-SST (sucrose:sucrose 1-fructosyltransferase) catalyses the first step in fructan biosynthesis while the remaining enzymes elongate the growing fructose chain (1-FFT: fructan:fructan 1-fructosyltransferase, 6G-FFT: 6-glucose fructosyltransferase, and 6-SFT: sucrose:fructose 6-fructosyltransferase). The enzymes 1-FEH or 6-FEH (fructoexohydrolase) reduce fructan chain length by releasing fructose molecules.
Bacteria use only one FT enzyme which contains both 1-SST and 1-FFT activities to synthesize high molecular weight fructan polymers. Most bacterial fructosyltransferases produce levan type fructan (levansucrases), which is characterized by β-2,6 linkages of fructose molecules, although inulosucrases that produce fructans of the inulin type (β-2,1 linkage) have been isolated from a few bacteria.
At least 3 bacterial levansucrases have been expressed in transgenic plants including, the SacB gene from Bacillus subtilis, the SacB gene from Bacillus amyloliquefaciens, and the FTF gene from Streptococcus mutans. Expression of these bacterial levansucrases in plants leads to the synthesis of very high molecular weight fructans of a DP of several thousands (for review see Cairns, 2003).
Fructans represent the major non-structural carbohydrate in 15% of plant species and play a key role in forage quality. Ruminant livestock grazing on high fructan diets show improved animal performance.
In grasses the level and composition of fructans has been increased in stems and leaf sheaths through the engineered expression of FT genes.
However, manipulating biochemical pathways by manipulating the activity of enzymes in the pathways may be difficult because of the ways in which the various enzymes and their substrates may interact.
Thus, it would be desirable to have improved methods of manipulating biochemical pathways, particularly in plants. For example, it would be desirable to have methods of manipulating fructan biosynthesis in plants, including forage grass species such as Lolium, Festuca, and Brachiaria; sugarcane and other grasses; and sorghum and other cereals such as wheat and maize; thereby facilitating the production of, for example:                forage grasses with improved herbage quality and/or yield, leading to improved pasture production, improved animal production and/or reduced environmental pollution;        bioenergy grasses and crops such as switchgrass, Miscanthus, sorghum (grain, forage and energy sorghum), sugarcane and energy cane with enhanced biomass yield, such as for bioethanol production; and        cereals such as wheat, rice, maize, barley with increased grain and/or biomass yield.        
Nucleic acid sequences encoding some of the enzymes involved in the fructan biosynthetic pathway have been isolated for certain species of plants. For example, PCT/AU01/00705 to the present applicants, describes fructosyltransferase homologues from Lolium and Festuca. However, there remains a need for materials useful in the modification of fructan biosynthesis in plants, and also to engineer fructan accumulation in different parts of the plant.
U.S. provisional patent application 61/097,008 describes constructs useful for modifying fructan biosynthesis in plants. However, those constructs preferably include a fusion gene encoding a translational fusion of two or more fructan biosynthetic enzymes, the genes making up the fusion preferably corresponding to plant fructan biosynthetic genes.
It is an object of the present invention to overcome, or at least alleviate, one or more of the difficulties or deficiencies associated with the prior art.